The present invention relates to methods for plant genetic transformation and for products thereof. More specifically, the present invention relates to the genetic transformation of any plant species with sexual reproduction based on a pollination-fecundation process. According to the present invention, pollen grains are pre-treated with silicon carbide fibers and the transforming DNA. The present invention also involves pollinating recipient plants with pollen grains carrying the transforming DNA.
Advances in molecular biology have dramatically expanded man""s ability to manipulate the germplasm of animals and plants. Genes controlling specific phenotypes, for example specific polypeptides that lend antibiotic or herbicide resistance, have been located within certain germplasm and isolated from it. Even more important has been the ability to take the genes which have been isolated from one organism and to introduce them into another organism. This transformation may be accomplished even where the recipient organism is from a different phylum, genus or species from that which donated the gene (heterologous transformation).
Genetic engineering of plants, which entails the isolation and manipulation of genetic material (usually in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant or plant cells, offers considerable promise to modern agriculture and plant breeding. Increased crop food values, higher yields, feed value, reduced production costs, pest resistance, stress tolerance, drought resistance, the production of pharmaceuticals, chemicals and biological molecules as well as other beneficial traits are all potentially achievable through genetic engineering techniques. Once a gene has been identified, cloned, and engineered, it is still necessary to introduce it into a plant of interest in such a manner that the resulting plant is both fertile and capable of passing the gene on to its progeny.
Developments in agrobiotechnology have resulted in a tremendous expansion of the capabilities for the genetic engineering of plants. Many transgenic dicotyledonous plant species have been obtained. However, many species of plants, especially those belonging to the Monocotyledonae and particularly the Gramineae, including economically important species such as corn, wheat and rice, have proved to be very recalcitrant to stable genetic transformation. Difficulties have been encountered in achieving both: a) integrative transformation of monocot plant cells with DNA (i.e., the stable insertion of DNA into the nuclear genome of the monocot plant cells) and b) regeneration from transformed cells of phenotypically normal monocot plants, such as phenotypically normal, fertile adult monocot plants. It has been suggested that such difficulties have been predominantly due to the nonavailability of monocot cells that are competent with respect to: 1) DNA uptake, 2) integrative transformation with the taken-up DNA, and 3) regeneration of phenotypically normal, monocot plants from the transformed cells (Potrykus (1990) Bio/Technology 9:535).
Thus, the introduction of exogenous DNA into monocotyledonous species and subsequent regeneration of transformed plants has proven much more difficult than transformation and regeneration in dicotyledonous plants. Moreover, reports of methods for the transformation of monocotyledons such as maize, and subsequent production of fertile maize plants, have not been forthcoming. Consequently, success has not been achieved in this area and commercial implementation of transformation by production of fertile transgenic plants has not been achieved. Thus there is a particularly great need for methods for improving genetic characteristics. Problems in the development of genetically transformed monocotyledonous species have arisen in many general areas. For example, there is generally a lack of methods, which allow one to introduce nucleic acids into cells and yet permit efficient cell culture and eventual regeneration of fertile plants.
Genetic engineering techniques have been successfully applied principally in dicotyledonous species. The uptake of new DNA by recipient plant cells has been accomplished by various means, including Agrobacterium infection (Nester, E. W., et al, (1984). Am. Rev. Plant Physiol 35: 387-413), polyethylene glycol (PEG) mediated DNA uptake (Lorz H., Baker B., Schell J. (1985). Mol Gen Genet 199:178-182.), electroporation of protoplasts (Fromm M. E., Taylor L. P., Walbot V. (1986). Nature 312:791-793.) and microprojectile bombardment (Klein T. M., Kornstein L., Sanford J. C., Fromm M. E. (1987). Nature 327: 70-73.).
The Agrobacterium transformation system is among the recombinant DNA technologies for genetic manipulation of plant genotypes. Virulent strains of the soil bacterium Agrobacterium turnefaciens are known to infect dicotyledonous plants and to elicit a neoplastic response in these plants. The tumor-inducing agent in the bacterium is a plasmid that functions by transferring some of its DNA into its host plant""s cells where it is integrated into the chromosomes of the host plant""s cells. This plasmid is called the Ti plasmid, and the virulence of the various strains of A. tumefaciens is determined in part by the vir region of the Ti plasmid which is responsible for mobilization and transfer of the T-DNA (Schell, J., Science, 237: 1176-1183 (1987)). The T-DNA section is delimited by two 23-base-pair repeats designated right border and left border, respectively. Any genetic information placed between these two border sequences may be mobilized and delivered to a susceptible host. Once incorporated into a chromosome, the T-DNA genes behave like normal dominant plant genes. They are stably maintained, expressed and sexually transmitted by transformed plants, and they are inherited in normal Mendelian fashion.
There are two common ways to transform plant cells with Agrobacterium: cultivation of Agrobacterium with cultured isolated protoplasts, or transformation of intact cells or tissues with Agrobacterium. The first requires an established culture system that allows for culturing protoplasts and subsequent plant regeneration from cultured protoplasts. The second method requires (a) that the intact plant tissues, such as cotyledons, can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
Agrobacterium-mediated transformation in dicotyledons facilitates the delivery of larger pieces of heterologous nucleic acid as compared with other transformation methods such as particle bombardment, electroporation, polyethylene glycol-mediated transformation methods, and the like. In addition, Agrobacterium-mediated transformation appears to result in relatively few gene rearrangements and more typically results in the integration of low numbers of gene copies into the plant chromosome.
However, the Agrobacterium transformation system, as stated, is restricted to certain dicot crops. For the majority of monocots, especially cereals (graminae) and grasses, A tumefaciens mediated gene transfer is not possible. Thus, the most important cultivated plants are not accessible for effective gene transfer.
A second frequently used process for transformation of plants is DNA direct delivery. One form of direct DNA delivery is direct gene transfer into protoplasts (using polyethyleneglycol treatment and/or electroporation). Protoplasts for use in such direct gene transfer methods have most often been obtained from embryogenic cell suspension cultures (Lazzeri and Lorz (1988) Advances in Cell Culture, Vol. 6, Academic press, p. 291; OziasAkins and Lorz (1984) Trends in Biotechnology 2: 1 19). However, the success of such methods has been limited due to the fact that regeneration of phenotypically normal plants from protoplasts has been difficult to achieve for most genotypes. For example, while regeneration of fertile corn plants from protoplasts has been reported, these reported methods have been limited to the use of non-transformed protoplasts. Moreover, regeneration of plants from protoplasts is a technique, which carries its own set of significant drawbacks. Even with vigorous attempts to achieve fertile, transformed maize plants, reports of success in this regard have not been forthcoming.
In yet another form of direct transformation, the genetic material is transferred using high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein, et al., Nature, 327:70-73 (1987)). In this method, non-biological particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. The main advantage of particle bombardment over Agrobacterium is absence of biological incompatibilities found when using this biological vector. In the plant kingdom, particle bombardment has shown good utility for transformation of conifers, dicots and monocots. However, particle bombardment has certain drawbacks relating to cost, ease of use, accessibility and end product utility. Moreover, transgenic plants obtained via Agrobacterium generally contain more predictable introduced DNA""s while partical bombardment, as well as other direct DNA uptake methods, give rise to more random and uncontrolled DNA integration events. Particle bombardment also often results in complex transgene insertion loci, which may cause gene silencing in some instances. In addition to their restrictive application in dicoytyledoneae and relatively low transformation rates, these systems require the regeneration of entire plants from plant protoplasts.
Thus, great difficulties remain also in employing methods of direct DNA delivery, due to high dependence on regeneration ability of the genotype. As a consequence, in the few known examples of successful transformation of maize the experimental material was based on the line A188 which is easy in regeneration. Noteworthy, in all of the methods based on multicellular target (embryos, leaf-discs or calli) the resulting transformed tissue is mosaic, demanding further steps to obtain non-mosaic progeny. Most of these difficulties are due to the use of long-term tissue culturing.
Another major problem in achieving successful monocot transformation has resulted from the lack of efficient marker gene systems, which have been employed to identify stably transformed cells. Marker gene systems are those, which allow the selection of, and/or screening for, expression products of DNA. For use as assays for transformed cells, the selectable or screenable products should be those from genetic constructs introduced into the recipient cells. Hence, such marker genes can be used to identify stable transformants.
Of the more commonly used marker gene systems are gene systems which confer resistance to aminoglycosides such as kanamycin. While kanamycin resistance has been used successfully in both rice (Yang et al., 1988) and corn protoplast systems (Rodes et al., 1988), it remains a very difficult selective agent to use in monocots due to high endogenous resistance (Hauptmann, et al., 1988). Many monocot species, maize, in particular, possess high endogenous levels of resistance to aminoglycosides. Consequently, this class of compounds cannot be used reproducibly to distinguish transformed from non-transformed tissue. New methods for reproducible selection of or screening for transformed plant cells are therefore needed. Accordingly, it is clear that improved methods and/or approaches to the genetic transformation of monocotyledonous species would represent a great advance in the art. Furthermore, it would be of particular significance to provide novel approaches to monocot transformation, such as transformation of maize cells, which would allow for the production of stably transformed, fertile corn plants and progeny into which desired exogenous genes have been introduced. The identification of marker gene systems applicable to monocot systems such as maize would provide a useful means for applying such techniques generally. The development of these and other techniques for the preparation of stable genetically transformed monocots such as maize could potentially revolutionize approaches to monocot breeding.
In order to overcome the difficulties of genotype-dependant transformation caused by low regeneration potential of cereals, many efforts were put to develop an alternative, genotype-independent transformation approach based on the pollination pathway (Ohta Y., 1986). In maize, high efficiency genetic transformation can be achieved by a mixture of pollen and exogenous DNA. (Luo Z. X. and Wu R., 1988, Proc. Natl. Acad. Sci. USA 83:715-719). Maize, often referred to as corn in the United Stated, can be bred by both self-pollination and cross-pollination techniques. Maize has separate male and female flowers on the same plant, located on the tassel and the ear, respectively. Natural pollination occurs in maize when wind blows pollen from the tassels to the silks that protrude from the tops of the ears. Transformation of rice via the pollen-tube pathway has also been demonstrated (Plant Molecular Biology Reporter 6:165-174). The major potential advantages of the pollen-tube pathway approach include: (a) genotype independence; (b) lack of mosaicism; (c) no need for complicated cell and tissue culture techniques.
Despite the keen interest in an effective transformation method having such advantages, no serious results have been obtained with this approach, because of low reproducibility. Nevertheless, partial transfer of alien genes into intact plants via pollination pathway has been reported in maize, tomato and melon (Chesnokov, Yu. V., et al, 1992, USSR Patent No. 1708849; Bulletin of the USSR Patents, No. 4; Chesnokov Yu. V. and Korol A. B. 1993; Genetika USSR, 29:1345-1355).
The procedures of genetic transformation based on the pollination-fecundation pathway include: (i) employment of a mixture (paste) of the pollen and transforming DNA; (ii) delivery of the alien DNA into the pollen tube, after pollination; and (iii) microparticle bombardment of microspores or pollen grains. The obstacles in application of the so-far developed versions of the pollination pathway of genetic transformation include: (i) very low reproducibility; (ii) extremely poor applicability to maize due to the very long style of this plant; and (iii) high cost (Potrykus, I. 1990. Gene transfer to cereals: an assessment. Bio/Technology 8:535-542). The present invention provides an alternative highly efficient method of plant genetic transformation and in particular of maize genetic transformation employing pollen treatment with silicon carbide fibers in the presence of foreign DNA.
Silicon carbide fiber technique has been used in plant genetic transformation procedures based on tissue culturing (Kaeppler, H. F., Somers, D. A., Rifles, H. W. and Cockburn, A. F. 1992. Silicon carbide fiberxe2x80x94mediated stable transformation of plant cells.
Theor. Appl. Genet. 84:560-566). Such an approach, is restricted by low regeneration potential of cereals in general, and maize in particular, limiting its application to elite cultivars. Moreover, this method provides only about 10% of the efficiency achieved by microparticle bombardment of the embryogenic tissues.
The present invention combines an improved process of pollination pathway and silicon fiber treatment that permits solving the above mentioned problems by delivering the transforming DNA into pollen grains and then, via the sperm, into the egg cells. This novel and non-obvious solution allows to achieve high frequency of maize transformation, and in other crops as well. Beside high efficiency and low cost, its most important advantages are high reproducibility, genotype independence, genetic stability of the transformants, and technical simplicity. These features, taken together, comprise a novel combination which allows said invention to become a basis for large-scale genetic transformation, especially in maize, but in other crops as well. The uniqueness of combining the pollination pathway and the delivering of the transforming DNA into pollen grains by silicon carbide fibers is that the method takes advantages of the natural reproduction system resulting in transformed zygotes.
The advantages of the developed strategy include: (1) expensive and time-consuming tissue culture techniques are not required, (2) genotype-independence, since the method does not require in vitro regeneration procedures, (3) elimination of plant sectoring (mosaicism), since the transformants result from zygotes, (4) no somaclonal variation and reduced fertility caused by prolonged tissue culturing, (5) the use of natural delivery system ensures high stability of the integrated DNA, (6) potential to transfer large fragments of alien DNA into the plant genome; and (7) low cost, high frequency and reproducibility.
Another important advantage of the present method is the possibility of using it for plant transformation (primarily cereals) by large fragments of DNA, e.g. cloned in yeast artificial chromosomes. This allows an increase in the efficiency of map-based cloning of genes of agronomical importance.
A method for plant transformation with resistant properties against antibiotics, herbicides as well as enhanced anthocyanin is provided.
The present invention is directed to a method for genetic transformation of any plant species with sexual reproduction based on a pollination-fecundation process, and its products thereof. According to the present invention the recipient plants are pollinated by pollen grains carrying the transforming DNA wherein the pollen grains are pre-treated by silicon carbide fibers and the transforming DNA. Accordingly, the present invention provides an improved process which combining the pollination pathway and the delivery of the transforming DNA into pollen grains by silicon carbide fibers. The method also allows the possibility to conduct controlled crosses.
The invention, more specifically, provides a method for plant transformation comprising pollination pathway and silicon fiber treatment such that the delivery of transforming DNA into pollen grains. The invention provides a novel and non-obvious process that allows high frequency of maize transformation, and in other crops as well. Beside high efficiency and low cost, its most important advantages and high reproducibility, genotype independence, genetic stability of the transformants, and technical simplicity. The invention further provides a method combining the pollination pathway and the delivering of the transforming DNA into pollen grains by silicon carbide fibers, which takes advantage of the natural reproduction system resulting in transformed zygotes.
The invention provides transgenic plants of the above-described method.
The invention also provides a paste comprising mixing silicon carbide fibers, pollen germination medium and DNA molecules.
Further objects and advantages of the present invention will be clear from the description that follows.